The present invention relates to a biosensor for facilitating simple and high accuracy quantitation of a substrate and a method for quantitative measurement of a substrate using the same.
There has been a conventional simple method for quantitating a specific component in a sample solution with no dilution or agitation of the sample solution. This method causes the specific component to react with an oxidoreductase whose substrate corresponds to the specific component in the presence of an electron mediator or electron acceptor and subsequently oxidizes the electron mediator reduced by the enzyme reaction electrochemically. The concentration of the specific component is then determined from the oxidation current flowing during this electrochemical oxidation.
This method normally uses a biosensor as disclosed in Japanese Laid-Open Patent Publication No. 2,517,153.
The biosensor is produced by first forming an electrode system having a working electrode and a counter electrode on an electrically insulating base plate by a screen printing method or the like, subsequently forming a reaction layer including an oxidoreductase and an electron mediator on the electrode system, and finally bonding a cover and a spacer to the electrically insulating base plate.
This biosensor facilitates quantitation of various specific components by varying the oxidoreductase.
A glucose sensor will be described as an example of biosensor.
Conventionally known method for quantitative measurement of glucose is a system comprising a combination of glucose oxidase with an oxygen electrode or a hydrogen peroxide electrode (e.g., "Biosensor", ed. by Shuichi Suzuki, Kodansha, Japan).
Glucose oxidase selectively oxidizes a substrate .beta.-D-glucose to D-glucono-.delta.-lactone by utilizing oxygen dissolved in a sample solution as an electron mediator. When the substrate is oxidized by the glucose oxidase, the oxygen used as the electron mediator is reduced to hydrogen peroxide. The glucose concentration can be quantitated either by measurement of the volume of oxygen consumed during this reaction using an oxygen electrode or by measurement of the volume of hydrogen peroxide produced using a hydrogen peroxide electrode of platinum or the like.
However, this method has a drawback that the measurement is largely affected by the concentration of oxygen contained in a sample solution, depending on the measuring object. This system has another drawback that the system cannot function in the absence of oxygen.
To overcome these problems, a type of glucose sensor has been developed which includes an organic compound or a metal complex such as potassium ferricyanide, ferrocene derivatives, quinone derivatives, etc. as electron mediator, in place of oxygen.
This biosensor can carry a known amount of glucose oxidase on an electrode system, together with an electron mediator in their stabilized state. This structure enables to combine the electrode system with the reaction layer integrally almost in dry state.
Such biosensor is normally disposable and facilitates measurement of the concentration of glucose by a simple instillation of a measuring sample at a sensor chip mounted in a measurement device. Therefore, this biosensor has been attracting much attention recently.
As described above, the substrate in a sample can be quantitated based on the current required for oxidizing the reductant of the electron mediator which has been produced by a series of enzyme reaction on the electrode.
However, some samples are assumed to contain an easy-to-oxidize substance which is oxidized upon oxidation of an electron mediator in reduced state on the electrode to generate an oxidation current and hence produces a positive error in the current value measured. Moreover, if the measuring sample contains a high concentration of substrate, the oxidation current value may vary.
In order to solve these problems, the present inventors propose a method for quantitative measurement of a substrate based on the reduction current value which flows during reduction of the electron mediator in oxidized state on the working electrode which remains not reduced by a series of enzyme reaction. This method precludes adverse effects of any easy-to-oxidize substance on the measurement results.
However, if the reduction current value is measured with a two-electrode system comprising a working electrode and a counter electrode, then the presence of some amount of electron mediator in reduced state which must be oxidized on the counter electrode in correspondence with the reduction of already oxidized electron mediator on the working electrode becomes mandatory. With this system, however, if the substrate concentration is predicted to be low, the electron mediator in reduced state may be depleted, which renders the oxidation on the counter electrode to develop a rate-determining step that adversely affects the reduction current value measured.